Bifunctional chimeric molecules for labeling of kinases with target binding moieties and methods of use thereof

Abstract

The present disclosure relates to chimeric small molecules, which find utility as modifiers of target substrates according to the formula A-L1-E-B or A-L1-E-L2-B, wherein A is a kinase binding moiety; B is a target binding moiety; L1 and L2 are each a linker; and E is an electrophilic reactive group. Molecules according to the present invention find use making substrate modifications such as post-translational modifications to targets that are not the natural substrate of the kinase; accordingly, diseases or disorders may be treated or prevented with molecules of the present disclosure.

What is claimed is: 
     
         1 . A chimeric small molecule according to the formula
   A-L 1 -E-B or A-L 1 -E-L 2 -B,   wherein A is a kinase binding moiety;   B is a target binding moiety;   L 1  and L 2  are each a linker; and   E is an electrophilic reactive group.   
     
     
         2 . The chimeric small molecule of  claim 1 , wherein the kinase binding moiety is an ABL, AMPK, PKC, FKBP, IRTK, SRC, Lyn, PK, NOP, e.g. GPC family for example; μ opioid receptor; or δ opioid receptor, MAPK, e.g. MAPK1; MAPK11; MAPK12; MAPK13; MAPK14; p38α MAPK, EGFR, BCKDK, FGFR, NGFR, IκB, CDK, e.g. CDK2; CDK4; CDK8, PI3K, VEGFR, BRAF, MEK, e.g. MEK1/2; MEK5, AKT, ALK, BTK, FLT3, JAK2, JAK3, AURKA, c-MET, DDR, INSR, IKK, mTOR, e.g. mTORC-1, PAK, PDK, e.g. PDK1 or PDK2, PTK2/FAK, pyruvate kinases, RAC-α, RIPK, TYK2, SHP, aPKC, e.g. PKC-ζ, SphK, GSK-3 binding moiety, UMPK, JNK, TrkA, PDGFR, ITK, IRE1 or LIMK. 
     
     
         3 . The chimeric small molecule of  any of the preceding claims , wherein the kinase binding moiety has a half-life shorter than the half-life of the kinase. 
     
     
         4 . The chimeric small molecule of  claim 3 , wherein the kinase binding moiety half-life is at least 2, 3, 4, 5 times shorter than the half-life of the kinase. 
     
     
         5 . The chimeric small molecule of  any of the preceding claims , wherein the kinase binding moiety is a kinase inhibitor or kinase activator. 
     
     
         6 . The chimeric small molecule of  any of the preceding claims , wherein the kinase inhibitor is a promiscuous kinase inhibitor. 
     
     
         7 . The chimeric small molecule of any of the preceding, wherein the electrophilic reactive group is selected from N-acyl-N-alkyl sulfonamide (NASA), dibromophenyl benzoate, or N-sulfonyl pyridone. 
     
     
         8 . The chimeric small molecule of  any of the preceding claims , wherein the electrophilic reactive group is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The chimeric small molecule of  any of the preceding claims , wherein the electrophilic reactive group reacts with a nucleophilic reactive group. 
     
     
         10 . The chimeric small molecule of  claim 1 , wherein the electrophilic reactive group has the formula: 
       
         
           
           
               
               
           
         
       
       wherein R 1  is selected from the group consisting of C—O, SO 2 , Me-C—O, and Me-SO 2 , R 2  is selected from the group consisting of H, alkane, alkene, alkyne, amine, nitrile, nitro, ether, alcohol, thiol, sulfone, sulfonate, halogen, carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide, cyclic hydrocarbon, an unsaturated cyclic hydrocarbon, a heterocycle, one or more fused rings thereof; an aliphatic halide such as —OCF 2 Cl and any combination thereof, and the benzene ring is optionally substituted at any position. 
     
     
         11 . The chimeric small molecule of  claim 10 , wherein the electrophilic reactive group is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         12 . The chimeric small molecule of  any of the preceding claims , wherein the electrophilic reactive group reacts with a nucleophilic group of one of Cysteine, Serine, Threonine, Tyrosine, Glutamic Acid, Aspartic Acid, Lysine, Arginine, and Histidine. 
     
     
         13 . The chimeric small molecule of  any of the previous claims , wherein L selected from the group consisting of: alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG, and any combination thereof. 
     
     
         14 . The chimeric small molecule  any of the previous claims , wherein L1 and L 2  are the same or are different molecules selected from the group consisting of alkane; alkene; alkyne; amine; ether; thiol; sulfone; carbonyl; acyl; ketone; carboxylate ester; amide; enone; anhydride; imide; PEG, and any combination thereof. 
     
     
         15 . The chimeric small molecule of  any of the preceding claims , wherein the kinase binding moiety further comprises a bio-orthogonal group. 
     
     
         16 . The chimeric small molecule of  any of the preceding claims , wherein the bio-orthogonal group is selected from tetrazines, triazines, cyclooctenes, cyclopropenes and diazo. 
     
     
         17 . The chimeric small molecule of  any of the preceding claims , wherein the bio-orthogonal group is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         18 . The chimeric small molecule of  any of the preceding claims , wherein the target is a protein. 
     
     
         19 . The chimeric small molecule of  claim 18 , wherein the target protein is from a pathogen 
     
     
         20 . The chimeric small molecule of  claim 19 , wherein the pathogen is a virus, bacteria, fungi, or protozoa. 
     
     
         21 . The chimeric small molecule of  claim 19 , wherein the microbial protein is in an intracellular or extracellular pathogen protein. 
     
     
         22 . The chimeric small molecule of  claim 21 , wherein the intracellular pathogen is  Mycobacterium tuberculosis  or the extracellular pathogen is  Pseudomonas aeruginosa.    
     
     
         23 . The chimeric small molecule of  claim 21 , wherein the kinase binding moiety is a phosphatase A (PtpA) binding moiety, PtpB binding moiety. 
     
     
         24 . The chimeric small molecule of  any of the preceding claims , wherein the chimeric small molecule is capable of covalently labeling a kinase with the kinase binding moiety. 
     
     
         25 . The molecule of  claim 24 , wherein the labeling is of a nucleophile disposed on the kinase. 
     
     
         26 . The chimeric small molecule of  claim 1 , wherein the target is capable of binding an oncogenic target. 
     
     
         27 . The chimeric small molecule of  claim 5 , wherein the kinase inhibitor is sorafenib, SB2035890 or Skepinone B, or an analog or derivative thereof. 
     
     
         28 . The chimeric small molecule of  claim 5 , wherein the kinase inhibitor is Gefitinib, or an analog or derivative thereof. 
     
     
         29 . The chimeric small molecule of  claim 5 , wherein the kinase inhibitor is Imatinib, or an analog or derivative thereof. 
     
     
         30 . The chimeric small molecule of  claim 5 , wherein the kinase inhibitor is Idelasilib, or an analog or derivative thereof. 
     
     
         31 . The chimeric small molecule of  claim 1  with the formula: 
       
         
           
           
               
               
           
         
         wherein m=0 or 1; n=1, 2, 3, 4 or 5; and X=CH 2 , O, or (CH 2 ) 2 O. 
       
     
     
         32 . The chimeric small molecule of  claim 1  with the formula: 
       
         
           
           
               
               
           
         
         wherein X and Y are independently selected from CH 2 , O, or (CH 2 ) 2 O and n and m are independently selected from 1, 2, 3, 4, 5, or 6. 
       
     
     
         33 . The chimeric small molecule of  claim 1  with the formula: 
       
         
           
           
               
               
           
         
       
     
     
         34 . A method of inducing modification of a target substrate comprising administering to a cell or cell population a chimeric small molecule of  any one of the preceding claims . 
     
     
         35 . A method of modifying a substrate comprising introducing a molecule of  any one of the preceding claims  to a cell. 
     
     
         36 . A method of modifying a target substrate in a cell, comprising generating a reprogrammed cellular kinase by delivering a chimeric small molecule of the formula
   A-L-E-B or A-L 1 -E-L 2 -B,   wherein A is a kinase binding moiety specific for the cellular kinase to be repurposed/reprogrammed;
 B is a target binding moiety specific for the target substrate to be modified; 
 L is a linker; and 
 El is an electrophilic reactive group 
   whereby the chimeric small molecule labels the cellular kinase with the target binding moiety for the target substrate; and   modifying the target substrate by binding of the repurposed/reprogrammed kinase to the target substrate via the target binding moiety, whereby the repurposed/reprogrammed cellular kinase introduces one or more modifications to the target substrate.   
     
     
         37 . The method of  claim 36 , wherein the kinase binding moiety is an ABL, AMPK, PKC, FKBP, IRTK, SRC, Lyn, PK, NOP, e.g. GPC family for example; μ opioid receptor; or δ opioid receptor, MAPK, e.g. MAPK1; MAPK11; MAPK12; MAPK13; MAPK14; p38α MAPK, EGFR, BCKDK, FGFR, NGFR, IκB, CDK, e.g. CDK2; CDK4; CDK8, PI3K, VEGFR, BRAF, MEK, e.g. MEK1/2; MEK5, AKT, ALK, BTK, FLT3, JAK2, JAK3, AURKA, c-MET, DDR, INSR, IKK, mTOR, e.g. mTORC-1, PAK, PDK, e.g. PDK1 or PDK2, PTK2/FAK, pyruvate kinases, RAC-α, RIPK, TYK2, SHP, aPKC, e.g. PKC-ζ, SphK, GSK-3 binding moiety, UMPK, JNK, TrkA, PDGFR, ITK, IRE1 or LIMK. 
     
     
         38 . The chimeric small molecule of any of  claims 36-37 , wherein the kinase binding moiety has a half-life shorter than the half-life of the target to which the target binding moiety is capable of binding. 
     
     
         39 . The method of  claim 36 , further comprising administering a coupling molecule thereby quenching the inhibitory activity of the kinase inhibitor. 
     
     
         40 . The method of  claim 36 , wherein modifying comprises inducing post-translational modification of a target protein. 
     
     
         41 . The method of  claim 40 , wherein the post-translational modification is phosphorylation. 
     
     
         42 . A method of treating cancer comprising generating a reprogrammed cellular kinase by administering to a subject in need thereof a chimeric small molecule of the formula:
   A-L-E-B, A-L 1 -E-L 2 -B, or A-(L) n -B   wherein A is a kinase binding moiety; L is a linker and n is between 0-6; E is an electrophilic reactive group and B is an oncogenic protein to be modified, whereby the chimeric small molecule labels the cellular kinase with the target binding moiety for the target substrate; and   modifying the oncogenic protein by binding of the repurposed/reprogrammed kinase to the target substrate via the target binding moiety, whereby the repurposed/reprogrammed cellular kinase introduces one or more modifications to the target substrate.   
     
     
         43 . The method of  claim 42 , wherein the kinase binding moiety is an inhibitor. 
     
     
         44 . The method of  claim 43 , wherein the kinase binding moiety is an ABL, AMPK, PKC, FKBP, IRTK, SRC, Lyn, PK, NOP, e.g. GPC family for example; μ opioid receptor; or δ opioid receptor, MAPK, e.g. MAPK1; MAPK11; MAPK12; MAPK13; MAPK14; p38α MAPK, EGFR, BCKDK, FGFR, NGFR, IκB, CDK, e.g. CDK2; CDK4; CDK8, PI3K, VEGFR, BRAF, MEK, e.g. MEK1/2; MEK5, AKT, ALK, BTK, FLT3, JAK2, JAK3, AURKA, c-MET, DDR, INSR, IKK, mTOR, e.g. mTORC-1, PAK, PDK, e.g. PDK1 or PDK2, PTK2/FAK, pyruvate kinases, RAC-α, RIPK, TYK2, SHP, aPKC, e.g. PKC-ζ, SphK, GSK-3 binding moiety, UMPK, JNK, TrkA, PDGFR, ITK, IRE1 or LIMK. 
     
     
         45 . The chimeric small molecule of any of  claims 43-44 , wherein the kinase binding moiety has a half-life shorter than the half-life of the kinase. 
     
     
         46 . The method of  claim 43 , further comprising administering a quenching molecule thereby quenching the inhibitory activity of the kinase inhibitor. 
     
     
         47 . A method for treating infection by a pathogen comprising:
 generating a reprogrammed cellular kinase by administering to a subject in need thereof a chimeric small molecule of the formula:
   A-L-E-B or A-L 1 -E-L 2 -B, 
   wherein A is a kinase binding moiety; L is a linker; E is an electrophilic reactive group and B is a pathogen protein to be modified, whereby the chimeric small molecule labels the cellular kinase with the target binding moiety for the target substrate; and   modifying the pathogen protein by binding of the repurposed/reprogrammed kinase to the pathogen protein via the target binding moiety, whereby the repurposed/reprogrammed cellular kinase introduces one or more modifications to the target substrate.   
     
     
         48 . The method of  claim 47 , wherein the kinase binding moiety is an ABL, AMPK, PKC, FKBP, IRTK, SRC, Lyn, PK, NOP, e.g. GPC family for example; μ opioid receptor; or δ opioid receptor, MAPK, e.g. MAPK1; MAPK11; MAPK12; MAPK13; MAPK14; p38α MAPK, EGFR, BCKDK, FGFR, NGFR, IκB, CDK, e.g. CDK2; CDK4; CDK8, PI3K, VEGFR, BRAF, MEK, e.g. MEK1/2; MEK5, AKT, ALK, BTK, FLT3, JAK2, JAK3, AURKA, c-MET, DDR, INSR, IKK, mTOR, e.g. mTORC-1, PAK, PDK, e.g. PDK1 or PDK2, PTK2/FAK, pyruvate kinases, RAC-α, RIPK, TYK2, SHP, aPKC, e.g. PKC-ζ, SphK, GSK-3 binding moiety, UMPK, JNK, TrkA, PDGFR, ITK, IRE1 or LIMK. 
     
     
         49 . The method of any of  claims 47-48 , wherein the kinase binding moiety has a half-life shorter than the half-life of the target to which the target binding moiety is capable of binding. 
     
     
         50 . The method of any of  claims 47 to 49 , wherein the kinase binding moiety is an inhibitor. 
     
     
         51 . The method of  claim 47 , further comprising administering a quenching molecule thereby quenching the inhibitor activity of the kinase inhibitor. 
     
     
         52 . The method of  claim 47 , wherein the pathogen is a virus, bacteria, fungi, or protozoa. 
     
     
         53 . The method of  claim 52 , wherein the bacteria is  Mycobacterium tuberculosis  (Mtb) or  Pseudomonas aeruginosa  (PsA). 
     
     
         54 . The method of  claim 53 , wherein the pathogen is Mtb and the pathogen protein is one or more of PtpA, PtpB, SapM, ESAT-6, and Rv2966c. 
     
     
         55 . The method of  claim 53 , wherein the pathogen is (PSA) and the target binding moiety is Colistin. 
     
     
         56 . The method of any of  claims 35 to 55 , wherein the electrophilic reactive group reacts with a nucleophilic group of one of Cysteine, Serine, Threonine, Tyrosine, Glutamic Acid, Aspartic Acid, Lysine, Arginine, and Histidine.